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AMYLOIDOSIS PRODUCED BY INJECTIONS OF PROTEINS(*).

The production of experimental amyloidosis in mice by repeated intramuscular injections of sodium caseinate (nutrose), which was first described by Kuczynski,(1) yields new possibilities for studying this interesting pathologic process. Previous to Kuczynski's work, amyloidosis was generally considered as being due to protracted infections and intoxications. This was in accordance with observations in human pathology, and with the results of earlier experiments in which amyloid degeneration had been observed following the injections of living or dead bacteria, or of bacterial toxins (Bailey(2)).

In attempting to explain his findings, Kuczynski assumed that the flooding of the body with foreign proteins caused the formation of abnormal intermediary products of the disturbed protein metabolism. These products dissolved with difficulty, and were precipitated from the supersaturated tissue fluids, thus forming the amyloid substance.

Kuczynski states that many injections of nutrose are necessary to secure positive results. By means of injections of bacteria, amyloid degeneration in mice can be produced in a much shorter time than with the caseinate, of which at least forty injections are required (Kuczynski, Strasser(3)).

RELATION BETWEEN AMYLOID DEGENERATION AND NUMBER OF PROTEIN INJECTIONS

In order to obtain exact information as to the number of injections that lead to the process of amyloidosis, a series of mice received daily injections with different amounts (0.2 to 1.0 cc.) of nutrose (Pfannstiehl). A 3 per cent. solution in physiologic sodium chlorid was prepared freshly every day, sterilized by boiling for five minutes and injected as sterile as possible alternately into the muscles of the left and right hind leg. The local reactions were slight. Aerobic and anaerobic cultures of the muscles were made after the animals had been killed, and these always remained sterile.

The results of these experiments are summarized in the table.

[TABULAR DATA NOT REPRODUCIBLE IN ASCII]

It appears from this table that certain individual differences exist regarding the disposition to amyloid degeneration when less than sixty injections are given. After about sixty injections, the results become uniform, amyloidosis being found in all animals receiving injections. At that time the amyloidosis may still be confined to the spleen, and a general amyloid degeneration is not constantly observed until eighty injections have been given.

If Kuczynski is right in stating that amyloidoses results from the flooding of the body with the products of an abnormal cleavage of foreign protein, we should expect a dependence of the degenerative processes on the amount of protein that is injected. Larger amounts should hasten the development. This, however, is not true. When 1 c.c. of the solution is given (the largest amount which can be injected into the leg of a mouse without causing severe damage), the same number of injections are required as when 0.2 cc. is used.

The question arises as to what the causal relation might be between the number of injections and the tissue alterations that lead to the formation of the amyloid substance. Casein and its easily soluble sodium salt are primarily not toxic. They cause slight reactions when introduced parenterally for the first time. This is the reason why casein has frequently been used in foreign protein therapy. Also, the mice do not seem to be affected by the injections of the caseinate during the first four to eight weeks. They gain in weight, and do not differ from the normal controls.

CHANGES OF BODY WEIGHT

Between the fifth to tenth week, the weight suddenly starts to drop, and goes down gradually to a level below that at the beginning of the experiment. Some of the mice died after from sixty to eighty injections, some survived as many as ninety to one hundred.

The change in the behavior of the body weight is a reliable indication of the presence of amyloidosis. The beginning of the decrease in weight precedes the onset of the degenerative changes from one to two weeks. When a mouse was killed while its weight was still going up, or had remained unchanged for a few days, amyloidosis was absent. When a mouse was examined that had lost weight during the last seven to fourteen days, amyloidosis was always found.

These observations suggest that the reaction of the organism to the foreign protein changes during the course of the injections and the nutrose, harmless for a certain period of time, finally becomes injurious. The changes in the body reactions that follow repeated injections of foreign protein have been studied by many investigators. and special attention should be called, in this connection, to the work by Schittenhelm and Weichardt.

CHANGES IN BLOOD PICTURE

The microscopic examination of the blood supports this assumption. The mice develop a slight anemia after from forty to sixty injections. The number of the erythrocytes goes down from 7 1/2 millions to 5 millions; the hemoglobin decreases from 110 per cent. to 80 per cent.

Pentimalli(4) described a hypochromatic anemia with leukocytosis in chronic protein poisoning of rabbits. Leukocytosis was also present in the mice receiving injections with nutrose. The leukocytosis, however, showed no relation to the onset or development of the amyloid degeneration. During the first weeks the white cells went up from 8,000 to 15,000 or 18,000; later their number remained more or less stationary, and in only a few mice was it finally above 20,000. The leukocytosis was due to an increase of the neutrophil leukocytes. Whereas in normal mice from 10 to 15 per cent. of the leukocytes are neutrophil granulated, their percentage in the mice that had received the nutrose solution amounted to from 40 to 50 per cent. The lymphocytes showed a corresponding decrease. The monocytes increased from 5 per cent to 8 or 10 per cent. The eosinophil leukocytes were not changed (1 to 2 per cent). Myelocytes were absent, and granulocytes with ring-shaped nuclei were the only less mature cell types found.

CHANGES OF TEMPERATURE REACTION(5)

Interesting results were obtained in recording the changes of the body temperature caused by injections of the caseinate. The injection of nutrose into a normal mouse is followed by a rise in the temperature, amounting to from 1 1/2 to 2 1/2 F., which reaches the maximum after from two to four hours. During the next two to four hours the temperature returns to normal (99 to 101 F.). With the increase in the number of injections, the rise in temperature becomes less and less pronounced, and after six or seven weeks, when a drop in the body weight is noted, the nutrose does not cause the temperature to rise, but, on the contrary, a decrease in temperature is now noted. The decrease is slight, its average being 2 F. This type of temperature reaction persists. In some of the mice, especially in those that had received very many injections, the temperature was found to be continuously lower than normal. In the morning, even before the injections were given, the temperature was from 2 to 3 F. below normal. The injection was either followed by a further decrease to 95 F. or less, or the temperature remained almost unchanged. Examination of these mice revealed extensive general amyloidosis.

Decrease in the temperature, according to Pfeiffer, points toward the toxic action of abnormal split products of the body proteins. Destruction of body tissue resulting from burning, photodynamic action of light, injection of trypsin, etc, leads to a sudden drop in temperature, which continues until the death of the animal. Application of heat (warm-box) may save the injured animal (Pfeiffer(6)).

The decrease in temperature and the decline in body weight that occurs after repeated injections of nutrose seem to indicate that the caseinate stimulates an abnormal toxic cleavage of body tissues in the sensitized mouse. The effect of the single injections is slight, but it is the frequent repetition of the irritation that finally becomes harmful. For a long time it has been known that amyloidosis results from protracted destruction of body tissues. There is no fundamental difference as to pathogenesis between the amyloidosis produced by the nutrose injections and that encountered in chronic infections and intoxications, since in all these conditions an abnormal breakdown of tissue apparently takes place.

EXPERIMENTS WITH SERUM

The question may arise as to whether the amyloidosis is confined to the action of the nutrose or whether other proteins may have a similar effect. To decide this question, a second series of mice received injections with fresh sterile human serum. The injections were given in the same way as those of the caseinate. These mice also gained in weight at first. A decline was again noted after the fortieth to sixtieth injection. When killed before this decline had started, no amyloid was found. During the sixth to eighth week, however, amyloid appeared first in the spleen, then in the liver, kidneys, suprarenals, lymph glands, etc. The same findings were obtained by using animal serum.

The amyloidosis that occurs after long continued injections of nutrose, human serum, etc., can therefore be considered as the result of a chronic protein poisoning; and it is typical of the mouse, an animal especially disposed to this form of degeneration.

MORPHOLOGY

The amyloid is first found in the spleen. In the earliest stages, it surrounds the malpighian bodies as bands; later, the pulp may be completely replaced by a coarse framework of amyloid. The luminae of the sinuses still exist, but their endothelium has disappeared, and the small amount of blood which they contain borders on amyloid.

The second organ to be involved is the liver; then the kidneys, suprarenals, lymph glands and intestines become involved. The changes finally become so extensive that almost every tissue, except the skin, the central nervous system and the sympathetic ganglions, is affected. The amyloidosis is much more pronounced than that observed after injections of bacteria, or that caused by the growth of transplantable malignant tumors. Thus, the kidneys, after many injections of the protein, showed the characteristic macroscopic picture of the third stage of the amyloid nephrosis. Their surface was covered with coarse granules, consisting of distended tubuli. In the depressions between the granules. a complete amyloid transformation of the glomeruli, with atrophy of the tubuli and proliferation of the interstitium, was found.

In the pancreas, amyloid was present, especially about the capillaries of the islands. Two localizations will be described more in detail. because they have not so far been recorded in experimental amyloidosis. In the heart, amyloid is not only deposited about the vessels of the myocardium, but it is also found in the leaflets of the valves, mainly in that of the valvula mitralis. Here it is located near the auricular surface as a compact layer, which becomes paler toward the middle of the valve, and it stands out distinctly against the valvular tissue proper (fig. 1).

[Figure 1 ILLUSTRATION OMITTED]

In the ovaries, amyloid is observed in the walls of the vessels, and in the corpora lutea. The deposits are so extensive that the corpora lutea appear as circumscribed amyloid tumors (fig. 2). The lutein cells are shrunken, contain little lipoid, and their nuclei are pyknotic.

[Figure 2 ILLUSTRATION OMITTED]

The amyloid produced by protein injections gives the microchemical reactions typical of this substance. It is necessary, however, for the fresh tissues to be fixed with 85 per cent. alcohol. It has often been stated that in experimental amyloidosis the metachromasia with gentian or methyl-violet is the only reaction which is distinct, the characteristic coloring with iodin or iodin sulphuric acid being absent, or much less pronounced (Bailey). The freshly deposited amyloid, in particular, is said to react only with the anilin dyes (Schmidt,(7) Davidsohn, and others).

In the experiments with nutrose and serum, the first traces of amyloid give a reaction with iodin at a time when the metachromatic coloration with methyl-violet is still hardly visible. The amyloid, when treated with iodin, has, however, a different color than the amyloid in the human tissues. It stained pink, and this color persisted and never turned mahogany brown, which is typical of the human amyloid. The addition of sulphuric acid changed the pink color to grayish blue or a pale green.

In the sections stained with methyl-violet the amyloid appeared bright purple. When methyl-green was used, it stained reddish violet.

With Congo-red (Bennhold(8)), the amyloid took up only a pale pink, and the brilliant red of the human amyloid was never observed.

The crystalline structure of the amyloid that Kuczynski describes is characteristic. Crystal-like bristles of amyloid were first observed by Maximow(9) in the horse liver. Domagk(10) found needles of amyloid in the liver of mice that had received large amounts of bacteria cultures. Similar observations were made in mice with tumors (Kuczynski). In other animals and in human beings the amyloid does not show this morphology.

The formation of the finest needles by the amyloid substance is not restricted to the liver. The tufts of the amyloid needles are found in most of the organs affected. Fixing with Zenker-Helly solution and staining with Mallory's anilin-blue method brings out these structures most distinctly.

The first deposits of the amyloid appear as fine membraneous foils at the basal membranes of the capillaries and at the reticular fibers. Cloudy masses about these membranes later become visible, and the needles differentiate out of the cloudy masses. They tend to arrange themselves radially about a more compact center, formed by a nodular thickening of the membrane or by a small cluster of disintegrated cells.

The stars and whirls of crystalline amyloid occupy the space between capillary wall and liver cells (fig. 3).

[Figure 3 ILLUSTRATION OMITTED]

In the corpora lutea of the ovary, whirls of long needles are most distinct. In the spleen the needles are less discernible, because the peripheral parts of the brushlike trimmings of neighboring sinuses are interlaced with each other. In the kidneys, needles are seen on the inside, of the basal membranes of the tubuli recti. In the glomeruli, the amyloid has a longitudinal fibrillation. The suprarenals show glittering bristles between the cortical cells and the capillary walls. The finest and shortest crystals are found on the surface of the reticular fibers of the lymph glands.

The needles cause no active cellular reaction in the surroundings, except in the liver. In this organ, proliferation of the Kupffer cells is noted after the injections have been continued for a very long time (from 90 to 100). The cells invade the crystalline clusters, destroy their regular shape, breaking up the stars and tufts into irregular debris. Under the influence of the proliferating histiocytes, the amyloid passes away, and is finally completely dissolved. In other places the endothelial cells fuse together in large giant cells, from 35 to 50 cc. in diameter, which phagocyte the amyloid crystals. By intracellular digestion the crystals lose the specific staining qualities, become paler and smaller, until only shadow-like remnants are left (fig. 4).

[Figure 4 ILLUSTRATION OMITTED]

It is interesting to note that the amyloid in the liver is removed while the injections are continued, since the statement is usually made that the amyloid is resorbed only after the injury has been eliminated (Dantschakow,(11) Kuczvnski).

In the other organs, however, endothelial resolution of the amyloid takes place only after the injections have been discontinued. That indicates that a special functional activity in the reticulo-endothelial system should be ascribed to the Kupffer cells.

The microscopic examination in the area of injections did not reveal significant changes. Amyloid, especially, was not found. There were very small groups of degenerated leukocytes surrounded by strands of scar tissue containing blood pigment, and a great many mast cells. The muscle fibers between the scar tissue showed fruitless attempts at regeneration by the formation of multinucleated buds.

COMMENT

The observation that amyloidosis results from repeated injections of foreign proteins has been explained on the basis of an acquired hyper-sensitiveness to the substance injected, causing an abnormal cleavage of body tissue. The nature of the protein that is used is less important than the number of the injections. The allergic stage, which is reached after about forty injections, is evident from the different type of the temperature reaction which follows the parenteral administration of the protein and from the sudden drop in weight.

Many observations prove that sensitized cells may respond to a renewed specific irritation with exhaustion and disintegration. In the experiments under discussion, microscopic signs of cellular alterations are visible. In the spleen, for instance, the giant cells greatly increase in number during the first weeks of the injections, and later become degenerated. Their protoplasm is homogeneous or vacuolated. Their nuclei are pyknotic, and the cells finally break down to form irregular debris. The liver cells contain vacuoles, lipoid granules and hyaline droplets. They are sometimes diffusely hyalinic. The nuclei often appear enlarged, irregular and hyperchromatic, and the Kupffer cells are filled with fat droplets. These changes precede the amyloid degeneration.

The experiments of Domagk, who produced amyloidosis in mice by injecting intravenously large amounts of living or dead bacteria, also seem to indicate some relation between amyloid degeneration and allergic reactions. Domagk found that injections of bacteria into immunized mice were followed by an enormous phagocytosis of the bacteria through the endothelial cells of the lung, liver, etc., and by an almost immediate appearance of amyloid. In human pathology little attention has been given to a possible relation between an allergic stage acquired in the course of a chronic infection and the appearance of the amyloid substance, although amyloidosis is most frequently found in tuberculosis, in which disease the occurrence of allergic reactions has been especially emphasized.

Human cases of amyloidosis are occasionally observed in which the most careful examination reveals no distinct cause (Eppinger,(12) Freundlich,(13) Husten(14). There are no signs of tuberculosis, of chronic suppuration, of leukemia, of syphilis or of malignant tumors. Novak mentions chronic catarrhalic inflammation of the colon. Freundlich describes an extreme general icterus. Is it not possible that the cases of apparently genuine amyloidosis are similar to the experimental amyloidosis following protein injections, in that they represent the effect of a pathologic hypersensitiveness to foreign proteins resulting from an abnormal permeability of the impaired intestine, or from an insufficiency of the liver, the importance of which for protein metabolism is well known?

`The large deposits of amyloid in the corpora lutea of the ovary recall that in human pathology the amyloid often shows an affinity for areas of increased or abnormal cellular activity. Askanazy (15) says that the localization of the amyloid depends partly on the kind and intensity of the function of an organ. In. cases of goiter the adenomatous nodes of the thyroid are more affected than the rest of the gland (Schilder,(16) Ipland(17). Schmidt pointed out that the newly formed inflammatory tissue is especially involved in the process of amyloidosis.

In the human skin, amyloid is found in close connection with the sweat glands and sebaceous glands, which display a great functional activity (Schilder). Tumors which have originated in the matrix of the hair may reveal local amyloidosis (Mallory, personal communication). Amyloid in the stroma of tumors has been described by Askanazy (scirrhus of the breast) and by Schmidt (adenoma of cortex of suprarenal. McCutcheon (18) observed amyloidosis of the suprarenals in a case of hypernephroma of the kidney.

Amyloid, however, may also be found in places in which the cellular activity is insignificant, as in the valves of the heart.

The question regarding the origin of the amyloid has usually been discussed from a morphologic standpoint, because this substance is characterized by its microscopic qualities, while its chemical nature is still waiting for a definite solution. A reliable and relatively simple method to produce amyloid degeneration will undoubtedly stimulate and aid new investigations of this problem, for all explanations brought forward up to the present time are of merely theoretical interest, and none of them is entirely satisfactory.

If the parenchymatous cells of the organs in which amyloid is found are the source of it, it can hardly be explained why the different cells should produce the same substance. Domagk's observations point toward the endothelial cells as playing an important role; but no particular changes in the endothelial cells have been observed in the mice receiving injections with nutrose or serum, apart from the fatty degeneration of the Kupffer cells in the liver. Endothelial cells, however, are found active in removing amyloid.

The origin from collagenous tissue (Mallory(19)) is probable. Collagenous tissue is present everywhere in the body, and the amyloid is intimately associated with it. The fibroblasts cannot be essential, because in the liver amyloid is found in the perivascular spaces between portal capillaries and liver cells, in which fibroblasts are absent. The reticular, fibrillar and membranous differentiations of mesenchymatous origin become secondarily involved. The amyloid tends to accumulate about them, and the fibrils are visible for a long time, although embedded in large amounts of the abnormal substance. Finally, there remains only the material between the fibrils and the membranes.

The blood has been regarded as containing the substances of which amyloid is formed in the tissue spaces (Kuczynski). No characteristic changes of the blood seem to precede or accompany the amyloid degeneration. In human cases the protein content of the serum, for instance, is found diminished or increased (Koref(20)). Dresel(21) tested the serum of patients with amyloidosis for the content of chondroidin sulphuric acid. He found an increase, but this increase was not specific, and was also present in patients with renal diseases. Besides, the analyses of some investigators have shown that chondroidin sulphuric acid is not a constant part of amyloid (Hansen,(22) Eppinger(13)).

Examination of the blood of the mice receiving injections with proteins did not yield any striking results. Of course, only a limited number of tests can be made with such small quantities of blood, and objections may be raised against the methods employed. However, the fact should be stated that the protein concentration of the serum determined by Dr. Kathe Dewey by means of Pulfrich's refractometer showed no characteristic changes, and that the hydrogen ion concentration was within normal limits (7.38 to 7.4).

SUMMARY

Intramuscular injections of nutrose or serum, when given over a certain period of time, have proved to 1)e a reliable method for producing amyloid degeneration in mice.

The reactions of the mice to the foreign proteins is changed during the course of the experiment. Whereas the injections at first cause a slight rise in the body temperature, they are later followed by a decrease in the temperature. With the change of the temperature reaction, the weight of the animals starts dropping. From one to two weeks later the first traces of amyloid become visible in the spleen.

The conclusion has been drawn from these observations that the amyloidosis occurring after long continued injections of protein results from an acquired hypersensitiveness to the injected substance.

After more than seventy injections, the amyloidosis becomes most extensive, even the valves of the heart being affected. An endothelial resorbtion of the amyloid finally takes place in the liver, even though the injections are continued.

(*) From the Department of Pathology and Bacteriology, University of Illinois, College of Medicine, and the Uihlein Memorial Laboratory of the Grant Hospital of Chicago.

(1.) Kuczynski: Virchows Arch. f. path. Anat. 239:225, 1922; Klin. Wchnschr. 2:722, 2193, 1923.

(2.) Bailey: J. Exper. Med. 23:773, 1916.

(3.) Strasser: Ztschr. f. d. ges. exper. Med. 36:381, 1923.

(4.) Pentimalli: Klin. Wchnschr. 3:2090, 1925.

(5.) The rectal temperature was taken. In order to secure reliable results, it is necessary for the mice to become accustomed to this procedure, otherwise the temperature may change because the animal is frightened. The rectum must be emptied before inserting the thermometer, which is allowed to remain in the rectum for from two to three minutes. Mice with bronchopneumonia (evident from an abnormal temperature) must be discarded.

(6.) Pfeiffer, H.: Ztschr. f. d. ges. exper. Med. 29:46, 1922.

(7.) Schmidt, M. B.: Virchows Arch. f. path. Anat. 254:606, 1925.

(8.) Bennhold: Munchen. med. Wchnschr. 69:1537, 1922.

(9.) Maximow: Virchows Arch. f. path. Anat. 1115:353, 1898.

(10.) Domagk: Virchows Arch. f. path. Anat. 253:594, 1924.

(11.) Dantschakow: Virchows Arch. f. path. Anat. 187:1, 1907.

(12.) Eppinger: Biochem. Ztschr. 122:107, 1921.

(13.) Freundlich: Med. Ktin. 19:1622, 1923.

(14.) Husten: Virchows Arch. f. path. Anat. 248:450, 1924.

(15.) Askanazy: Beitr. z. path. Anat. u. z. allg. Path. 71:583, 1923.

(16.) Schilder: Beitr. z. path. Anat. u. z. allg. Path. 46:602, 1909.

(17.) Ipland: Frankfurter Ztschr. f. Path. 16:441, 1915.

(18.) McCutcheon: Am. J. M. Sc. 166:197, 1923.

(19.) Mallory: Pathologic Histology, Philadelphia, W. B. Saunders Company, 1914.

(20.) Koref: Med. Klin. 20:1243, 1924.

(21.) Dresel: Klin. Wchnschr. 2:2344, 1923.

(22.) Hansen: Biochem. Ztschr. 13:185, 1908.
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Author:JAFFE, RICHARD H.
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Jan 1, 2001
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